While the use of live bacteria in DNA vaccination and gene therapy has shown significant potential in cancer therapy, another avenue has been the use of bacterial products such as lipolysaccharides, peptidoglycans or even naked DNA. Vassaux et al., J. Pathol. 208:290-298 (2006). As early as in 1984 the DNA from Myobacterium bovis BCG was shown to have antitumor properties. Indeed, Tokunaga et al. described the isolation of purified DNA from BCG that demonstrated significant tumor regression in mice and substantial regressing response in human skin malignancies. Tokunaga et al., Japan J. Infect. Dis. 52:1-11 (1999).
The immunostimulatory activity leading to tumor regression in the BCG DNA is due to the presence of DNA stretches rich in unmethylated CpG dinucleotides. Unmethylated CpG sequences are 20 times more common in bacterial DNA as compared to mammalian DNA, and the presence of specific unmethylated CpG sequences (motifs) are recognized by the Toll-like receptor (TLR) 9. This interaction between the bacterial CpG DNA motif and TLR9 activates the monocytes and dendritic cells to produce interleukin-12 which in turn activates the T-helper 1 cells. In contrast to CpG DNA, bacterial lipopolysaccharides are recognized by TLR4 while the corresponding lipoproteins are recognized by TLR2. Modlin, Nature 408:659-660 (2000); Krieg, Nature Med. 9:831-835 (2003). Since the bacterial DNA rich in unmethylated CpG sequences is difficult to purify for human clinical trials, synthetic oligodeoxynucleotides (ODNs) of 8 to 30 bases and containing one or more CpG motifs, have been used with encouraging results in the immunotherapy of viral, bacterial and parasitic infections, as well as in limited phase I human clinical trials in patients with basal cell carcinoma or melanoma. Krieg, Id.
In contrast to bacterial CpG motifs, mammalian cell DNA contains CpG sequences where the cytosine residues are highly methylated. For example, about 6 to 8% of human DNA cytosine residues are believed to be methylated by DNA methyltransferases whose levels are modestly higher in human tumors than in normal cells. The promoter regions of a number of human genes have CpG islands that are hypermethylated leading to silencing of the downstream genes. Such epigenetic silencing of the downstream genes, particularly in tumor suppressor genes such as p16Ink4a, BRCA1 or hMLH1, triggers tumor formation and where development of specific DNA-demethylating agents can serve as antitumor drugs. Herman and Baylin, New Eng. J. Med. 349:2042-2054 (2003); Feinberg and Tycko, Nature Rev. Cancer 4:143-153 (2004). Transcriptional profiling of several breast cancer cell lines has demonstrated the presence of CpG islands that are highly methylated in the primary tumors and in the cell lines but not in the DNA from normal breast epithelia or matched lymphocytes from cancer patients. Treatment of a breast cancer cell line with the DNA methylation inhibitor 5-azacytidine resulted in the growth arrest of the cancer cells, demonstrating the importance of promoter CpG island methylation in tumor growth. Wang et al., Oncogene 24:2705-2714 (2005).
What is needed are new treatments for patients, specifically patients suffering from conditions, and specifically cancer, and preventative treatments for cancer. Such treatments may be new treatment methods, or variations or improvements on previously described treatment methods. Such cancer treatments should be able to slow the growth of tumors in mammalian patients, and/or decrease the size of tumors in mammalian patients. Also needed are methods to deliver therapeutic molecules to cancer cells in a patient as well as diagnose the existence and location of tumors in patients.